Recombinant Pongo abelii Nardilysin (NRD1), partial

What is Nardilysin (NRD1) and what are its primary functions in cellular processes?

Nardilysin (NRD1, now known as NRDC) is a zinc-dependent endopeptidase that belongs to the peptidase M16 family. It primarily cleaves peptide substrates at the N-terminus of arginine residues in dibasic moieties . At the subcellular level, NRD1 localizes to mitochondria where it functions as a co-chaperone for α-ketoglutarate dehydrogenase (OGDH), a rate-limiting enzyme in the Krebs cycle .

The protein has several critical functions:

• Acts as a mitochondrial co-chaperone assisting in the folding of OGDH

• Regulates cellular metabolism through influencing the Krebs cycle

• Participates in cell migration and proliferation via interaction with heparin-binding EGF-like growth factor

• Functions as a critical activator of BACE1- and ADAM17-mediated pro-neuregulin ectodomain shedding involved in axonal maturation and myelination

Methodologically, researchers should employ subcellular fractionation techniques when studying NRD1 to properly isolate and characterize its mitochondrial functions versus its cytosolic roles.

How does NRD1 contribute to neurodegeneration pathways?

NRD1 plays a crucial role in preventing neurodegeneration through several interconnected mechanisms:

• Mitochondrial quality control: NRD1 recruits mitochondrial chaperones and assists in the folding of OGDH, maintaining proper mitochondrial function .

• Metabolic regulation: Loss of Nrd1 leads to increased α-ketoglutarate levels (an OGDH substrate), which activates mTORC1 signaling .

• Autophagy modulation: The activated mTORC1 subsequently reduces autophagy, a process essential for clearing damaged cellular components .

• Neuroprotection: Inhibiting mTOR activity (using rapamycin) or partially restoring autophagy can delay neurodegeneration in Nrd1-deficient models .

This mechanistic pathway provides a direct link between mitochondrial metabolic dysfunction, mTORC1 signaling, and impaired autophagy in the context of neurodegeneration. Researchers studying recombinant Pongo abelii NRD1 should design experiments that assess each of these pathway components.

What expression systems are most appropriate for producing functional recombinant Pongo abelii NRD1?

When selecting an expression system for recombinant Pongo abelii NRD1, researchers should consider the protein's mitochondrial localization and complex functions. Based on general recombinant protein methodologies, recommended approaches include:

Given NRD1's role as a mitochondrial protein with chaperone function , mammalian or insect cell expression systems likely offer the best balance of proper folding and yield for producing functional protein.

How can researchers distinguish between the endopeptidase and chaperone functions of recombinant Pongo abelii NRD1?

NRD1 exhibits dual functionality as both an endopeptidase and a mitochondrial co-chaperone , requiring specialized methodological approaches to study each function independently:

• Structure-based mutagenesis:

  • Identify and mutate catalytic residues in the peptidase domain

  • Create point mutations in chaperone domains that preserve peptidase activity

  • Validate mutants with specific activity assays

• Domain-specific constructs:

  • Express the peptidase domain separately from chaperone regions

  • Assess each domain's activity independently

• Parallel functional assays:

  • Endopeptidase activity: Fluorogenic peptide substrates with arginine at P1 position

  • Chaperone activity: OGDH folding assays measuring prevention of aggregation

  • Thermal shift assays to assess protein stabilization effects

• Subcellular localization:

  • Mitochondrial isolation to assess chaperone function

  • Cytosolic fraction analysis for endopeptidase activity

These methodological approaches allow researchers to dissect and quantify each function independently, providing a more comprehensive understanding of NRD1's multifunctional nature.

What are the key considerations when investigating the effect of partial recombinant Pongo abelii NRD1 on mTORC1 signaling and autophagy?

When studying partial recombinant NRD1's effects on mTORC1 signaling and autophagy, researchers should implement a structured experimental approach:

• Domain mapping and structural analysis:

  • Identify which functional domains are present/absent in the partial protein

  • Use computational modeling to predict functional consequences

• Metabolic profiling:

  • Measure α-ketoglutarate levels, as NRD1 deficiency leads to its accumulation

  • Monitor OGDH activity to assess enzyme folding efficiency

• mTORC1 activation assessment:

  • Phosphorylation status of S6K1 and 4E-BP1 (direct mTORC1 targets)

  • Response to rapamycin treatment as positive control

• Autophagy measurement:

  • LC3-I to LC3-II conversion rates

  • p62 accumulation/degradation kinetics

  • Autophagic flux using tandem fluorescent reporters

• Comparative analysis:

  • Full-length versus partial NRD1 effects on each pathway component

  • Rescue experiments in NRD1-deficient cellular models

This comprehensive approach enables researchers to determine whether partial NRD1 retains sufficient functionality to regulate these critical pathways linked to neurodegeneration.

How do experimental conditions affect recombinant Pongo abelii NRD1 activity in mitochondrial function assays?

Mitochondrial function assays require careful optimization when studying recombinant NRD1:

Additionally, researchers should consider:

• Isolation methods for intact mitochondria that preserve NRD1-OGDH interactions

• Membrane permeabilization techniques that maintain mitochondrial structural integrity

• Oxygen consumption measurements to assess OGDH function in the electron transport chain

• ROS production monitoring to evaluate mitochondrial stress responses

These methodological considerations are essential for obtaining physiologically relevant data about NRD1's mitochondrial functions.

What strategies can overcome poor solubility and stability of recombinant Pongo abelii NRD1?

Solubility and stability challenges are common when expressing recombinant mitochondrial proteins like NRD1. Methodological approaches include:

• Expression optimization:

  • Reduce induction temperature (16-20°C)

  • Extend expression time (overnight)

  • Use specialized media formulations with osmolytes

• Fusion partners and tags:

  • SUMO tag for enhanced solubility

  • MBP fusion for improved folding

  • Split-GFP system for solubility monitoring

  • Consider tag position (N vs. C-terminal) based on mitochondrial targeting sequence location

• Buffer optimization matrix:

  • pH range: 6.5-8.0

  • Salt type and concentration: NaCl (50-500 mM)

  • Additives: Glycerol (5-20%), arginine (50-100 mM)

  • Detergents: Mild non-ionic (0.01-0.1%)

• Stability assessment:

  • Thermal shift assays to identify stabilizing conditions

  • Limited proteolysis to identify flexible regions

  • Size exclusion chromatography to monitor aggregation state

• Co-expression strategies:

  • Mitochondrial chaperones (HSP60/HSP10)

  • OGDH components to facilitate co-folding

Researchers should systematically document each approach's impact on yield, purity, and functionality of the recombinant protein.

How can researchers validate that recombinant Pongo abelii NRD1 retains native functionality?

Validating native functionality requires multiple complementary approaches:

• Endopeptidase activity assays:

  • Synthetic fluorogenic substrates containing dibasic motifs

  • Cleavage site specificity analysis using mass spectrometry

  • Inhibitor sensitivity profiling

• Mitochondrial chaperone function:

  • OGDH folding assistance measurement

  • Prevention of protein aggregation under stress conditions

  • Co-immunoprecipitation with known chaperone partners

• Cellular rescue experiments:

  • Complementation of NRD1-deficient cells

  • Restoration of OGDH activity

  • Normalization of α-ketoglutarate levels

  • Rescue of autophagy defects

• Structural integrity assessment:

  • Circular dichroism for secondary structure

  • Limited proteolysis for domain folding

  • Size exclusion chromatography for oligomeric state

• Comparative analysis:

  • Side-by-side comparison with human NRD1

  • Species-specific activity differences quantification

These validation steps ensure that experimental findings with the recombinant protein accurately reflect native NRD1 biology.

What statistical approaches are most appropriate for analyzing NRD1 enzymatic activity across experimental conditions?

Robust statistical analysis is essential for reliable interpretation of NRD1 enzymatic data:

• Experimental design considerations:

  • Minimum of three biological replicates

  • Technical triplicates within each biological replicate

  • Inclusion of appropriate positive and negative controls

  • Randomized sample processing order

• Kinetic parameter determination:

  • Non-linear regression for Michaelis-Menten kinetics

  • Global curve fitting for comparative analyses

  • Confidence interval calculation for all parameters

• Statistical tests for comparisons:

  • Paired t-tests for before/after treatments

  • One-way ANOVA with post-hoc tests for multiple conditions

  • Two-way ANOVA for interaction effects (e.g., pH × temperature)

• Advanced approaches for complex datasets:

  • Principal component analysis for multidimensional data

  • Linear mixed-effects models for nested experimental designs

  • Bootstrapping for robust parameter estimation

• Recommended data presentation format:

These approaches ensure rigorous and reproducible analysis of enzymatic data.

How should researchers interpret conflicting results between in vitro and cellular studies with recombinant Pongo abelii NRD1?

Discrepancies between in vitro and cellular findings are common in protein research. A systematic approach to resolving such conflicts includes:

• Protein characterization validation:

  • Confirm structural integrity in both systems

  • Verify post-translational modifications

  • Assess oligomerization state

• Environmental factors consideration:

  • pH differences between test tube and cellular compartments

  • Redox state variations

  • Presence of cellular binding partners

  • Availability of metal cofactors

• Concentration effects analysis:

  • Perform dose-response studies across wider ranges

  • Consider physiological versus experimental concentrations

  • Evaluate potential aggregation at higher concentrations

• Subcellular localization verification:

  • Confirm proper targeting to mitochondria in cellular systems

  • Fractionate cells to determine actual protein distribution

  • Use fluorescent tagging to visualize localization

• Methodological reconciliation:

  • Design hybrid experiments bridging in vitro and cellular approaches

  • Use permeabilized cell systems

  • Develop reconstituted systems with defined components

When documenting conflicting results, researchers should present both datasets with detailed experimental conditions to facilitate interpretation of the discrepancies.

How can recombinant Pongo abelii NRD1 be utilized in comparative models of neurodegeneration?

Recombinant Pongo abelii NRD1 offers unique opportunities for comparative neurodegeneration research:

• Evolutionary insights approach:

  • Compare protein sequence and structure between human and orangutan NRD1

  • Identify conserved versus divergent regions

  • Correlate with species-specific neurodegeneration susceptibility

• Cross-species rescue experiments:

  • Introduce Pongo abelii NRD1 into human neuronal models with NRD1 deficiency

  • Assess rescue efficiency compared to human NRD1

  • Identify potential protective mechanisms

• Metabolic pathway analysis:

  • Compare α-ketoglutarate metabolism regulation

  • Measure mTORC1 activation dynamics

  • Quantify autophagy modulation efficiency

• Mitochondrial function assessment:

  • Membrane potential maintenance

  • ROS production under stress conditions

  • ATP synthesis capacity

  • Mitochondrial morphology and dynamics

• Therapeutic target identification:

  • Screening for compounds that enhance NRD1 chaperone function

  • Comparative drug response between human and orangutan systems

  • Structure-activity relationship studies for precision therapeutics

This comparative approach may reveal evolutionary adaptations in NRD1 function that could inform novel therapeutic strategies for neurodegenerative disorders.

What methodological considerations are important when investigating the relationship between NRD1, mTORC1 signaling, and autophagy?

Research has established that NRD1 loss affects autophagy through mTORC1 signaling . When investigating this pathway, researchers should implement:

• Sequential pathway analysis:

  • OGDH folding and activity measurement

  • α-ketoglutarate level quantification

  • mTORC1 activation assessment

  • Autophagy flux determination

• Temporal resolution studies:

  • Time-course experiments after NRD1 addition/depletion

  • Pulse-chase approaches for metabolite tracking

  • Live-cell imaging with temporal resolution

• Pharmacological validation:

  • Rapamycin as positive control for mTOR inhibition

  • Torin1 for more complete mTORC1/2 inhibition

  • Bafilomycin A1 for autophagy flux assessment

• Genetic manipulation approaches:

  • CRISPR/Cas9-mediated NRD1 knockout/knockin

  • Inducible expression systems

  • Domain-specific mutants

• Quantitative data collection:

  • Phospho-specific western blotting for mTORC1 targets

  • Flow cytometry for autophagy markers

  • High-content imaging for spatial information

  • Metabolomics for comprehensive TCA cycle analysis

These methodological considerations ensure rigorous investigation of the NRD1-mTORC1-autophagy axis, providing mechanistic insights into neurodegeneration pathways that could be targeted therapeutically.